Recent data have linked two different rare mutations in the a synuclein gene with early onset familial Parkinson's disease. We and other have found a synuclein immunoreactivity to be present in Lewy bodies and other pathological lesions in the Parkinson disease brain, even in sporadic cases. These data suggest that a synuclein plays a critical role in the disease process. a Synuclein is predominantly expressed in neurons. Its function is unknown. This proposal will study a synuclein using three complementary systems: human brain samples, tranfected primary hippocampal or brain stem neurons, and over-expressing transgenic mice. We have developed novel applications of confocal scanning laser microscopy and fluorescence resonance energy transfer (FRET) to study the subcellular distribution of a synuclein and a synuclein-ubiquitin conjugates in brain. Transfection of primary neurons with Green Fluorescent Protein-alpha synuclein fusion constructs reveals a membrane association at presynaptic specializations in living neurons- application of FRET techniques to these neurons will be used to study a synuclein intramolecular interactions with membranes, and gain insight into a synuclein (and mutant a synuclein) conformation. Moreover, a synuclein GFP changes its location and merges with the membrane upon depolarization. We will study the impact of the mutations and of other stimuli such as MPP+ on this phenomenon using 2 photon confocal microscopy. We will also propose to generate transgenic mice with wild type and both mutant forms of a synuclein using the hamster PrP promoter which has previously been successful in other neurodegenerative disease models. We will build on the results of aims #1 and #2 to anatomically phenotype the mice, as well as relying on the resources of the Center to examine the mice for behavioral or electrophysiological (Dr. Graybiel) or neurochemical (Dr. Penny) abnormalities. Together, we propose an integrated, comprehensive approach to understanding the role of a synuclein in Lewy body diseases.